The present invention is directed to a method and apparatus for determining the level of fibrinogen in a blood sample. Specifically, the present invention is directed toward a method and microprocessor based apparatus for determining the level of fibrinogen in a sample using a piece-wise function which is generated empirically and pre-stored in a non-volatile memory.
The process of blood coagulation is complex. In general, it involves the generation of fibrin fibers which are formed by the polymerization of molecules of a protein called fibrinogen. Fibrinogen is catalyzed from an enzyme called thrombin, which is itself catalyzed from the enzyme prothrombin.
The prothrombin time test (PT test) is commonly used to determine the ability of a blood sample to clot. This test is extensively used in hospitals, clinics, and laboratories for pre-operative evaluations and for anti-coagulant therapy administered to cardiac patients, for example. The PT test is based upon the length of time required for a sample of plasma to clot under the influence of certain reagents. In the PT test, these reagents are calcium ion and thromboplastin. Thromboplastin is derived from the brain, lung, placenta and other tissues of humans, rabbits, cows, and other non-human species.
Methods for detecting the formation of fibrin clots date back to the late 1870's. Such early methods were manual. For example, in one test a white horse hair was drawn through a blood specimen. The end point of the clotting time was the point where shreds of fibrin could be visually detected on the hair.
By 1910, an electrical apparatus called a "Coaguloviscosimeter" was developed for directly measuring the change in viscosity of a blood sample as it clots. The apparatus provided a direct indication of voltage which could be plotted against clotting time.
In the early 1920's, rudimentary photoelectric techniques were developed to detect variations in light transmissivity of a blood sample during clotting. An apparatus termed a "Nephelometer" was devised. This device consisted of a light source which furnished a constant illumination to a sample. During coagulation of the sample, variations in the optical transmissivity of the sample were registered by a thermopile connected to a sensitive galvanometer. By reading the movement of the galvanometer needle, transmissivity values could be plotted against elapsed time.
In the mid-1930's, investigations of the coagulation of blood plasma using more sophisticated photoelectric techniques were conducted. It was noted that an increase in optical density occurs as blood coagulates. The possibility of detecting this change by photoelectric techniques was investigated. This lead to the development of an instrument which displayed increasing density of the same as a gradual change in the voltage displayed by a galvanometer. In addition, a water bath was used to maintain the blood sample at 37.degree. C.
Modern optical density detection systems operate on the principle that an increase in the optical density of a coagulating plasma sample will decrease the transmissivity of light through the sample. In a typical optical density detection system, a test blood plasma sample is placed in a transparent sample cuvette and reacted with a test reagent such as thromboplastin. Light or electro-magnetic radiation in the visible or near-infrared spectrum is then passed through the plasma-reagent mixture as the sample clots. As the biochemical change leading to fibrin formation takes place within the sample, the optical density of the sample increases. Output voltages corresponding to the optical density of the sample and the rate of change in optical density of the sample with respect to time are outputted, and used to determine coagulation end points.
While the existence of the relationship between fibrinogen (fibrin) levels and optical density has long been recognized, there has been wide disagreement concerning the nature and proper methodology for measuring the relationship, and numerous test parameters have been devised for determining fibrinogen levels using optical density data.
Several prior art techniques focused upon determining an optical "end point" corresponding to the initiation of fibrin formation. U.S. Pat. Nos. 3,658,490 and 3,307,392, for example, disclosed a method and apparatus which generated a signal corresponding to the value of the first differential of optical density with respect to time at the point corresponding to the incipiency of fibrin formation. In the method and apparatus of U.S. Pat. No. 3,458,287, the second differential of optical density with respect to time was measured to determine the maximum value of the first differential optical density, a value also corresponding to a preselected optical density time point. Each of these tests therefore measured an optical "end point" related to the rate of fibrin clot formation.
The test methods discussed above contained several flaws. Most notably, the rate of fibrin formation, while clinically significant, is not always indicative of the fibrinogen level of the sample. Rather, it may be a function of a given sample's ability to produce thrombin, the material which converts fibrinogen to fibrin. Two samples having equal levels of fibrin may produce different optical density test curves if each sample produces thrombin at a different rate.
Some prior art systems attempted to compensate for this fact by focusing on an absolute change in the optical density of a sample between a pre-clot or initial state and a preselected final state. While such systems eliminate the inaccuracies caused by factors such as differences in the thrombin levels between respective samples, they incorrectly assumed a uniformity in the initial optical densities of pre-clotted samples. Some blood samples, for example, may be cloudy due to the presence of lipid, which will alter the initial optical density of the sample. A system which solves some of these problems has been developed by Ortho Diagnostic Systems, Inc. of Raritan, N.J. The Ortho Diagnostic system adjusts the intensity of the light source between samples. However, the Ortho Diagnostics system does not provide a clinically acceptable parameter linking a coagulation end point or optical density data with fibrinogen levels.
In addition to the empirical problem of choosing clinically significant optical end points for determining the levels of fibrinogen using optical density data, prior art methods and apparatus further incorrectly assume that the relationship between changes in optical density and fibrinogen level is linear. See, U.S. Pat. No. 4,289,498. Prior art systems which assume a purely linear relationship between optical density change and fibrinogen levels have typically determined fibrinogen levels by constructing a best fit linear approximation using a plurality of optical density data points calculated from samples having fibrinogen levels within the normal range.
Linear approximation methods, while accurate predictors of fibrinogen levels in the normal range, are characterized by two basic problems. First, such methods typically do not produce best fit linear approximations which intercept both axes at the origin. In theory and practice, however, a plasma sample having a zero fibrinogen level cannot clot, and therefore cannot exhibit any change in optical density. This fact plainly suggests that the theoretical endpoint of any fibrinogen versus optical density time graph must be centered at the origin and raises doubts as to the accuracy of low fibrinogen levels calculated using linear best fit approximations.
Second, the linear relationship between change in optical density and fibrinogen levels also breaks down at fibrinogen levels significantly above the normal range. Most methods which utilize linear best fit approximations, utilize linear extrapolation above the normal, producing lines which suggest the theoretically impossible result of an optical density greater than complete opaqueness. Plainly, fibrinogen levels determined using conventional best fit linear approximations are not always accurate.
It is therefore the principal object of the method and microprocessor based apparatus of the present invention to resolve the problems associated with prothrombin time (PT) and other blood coagulation tests utilizing prior art optical density fibrinogen methods.
The present invention performs the PT test in association with a novel parameter designated as DELTA, which compensates for the inaccuracies associated with prior art optical density systems based on linear best fit approximations. DELTA in the present invention is defined as the ratio of an output voltage corresponding to the pre-clot optical transmissivity voltage level (PCOV) minus a special end of reaction term corresponding to a transmission voltage measured at the time when the rate of change in optical density decreases to one-half of its previous maximum value (ACOV), the difference being divided by (PCOV); EQU DELTA=(PCOV-ACOV)/PCOV
ACOV represents a point on the optical transmissivity time curve at which almost all clot formation activity is complete and remaining clot formation proceeds in a predictable fashion. Thus, differences in reaction rates between different blood plasma samples have minimal effect on the ACOV measurement. Further, because the difference of the numerator is divided by pre-clot voltage (PCOV), inaccuracies caused by differences in pre-clotting optical densities are factored out of the equation.
The relationship between the DELTA parameter and fibrinogen level is then directly determined from a piecewise function constructed using three DELTA points calculated from preselected reference samples having known low, normal and high fibrinogen levels which are stored in a non-volatile memory and used in association with one of four equations pre-stored in a non-volatile memory. The unknown fibrinogen level of a patient blood sample is calculated by the microprocessor using one of the four pre-stored equations which utilize the pre-stored reference fibrinogen values and their corresponding DELTA values.
The piece-wise function of the present invention provides accurate results for patient blood samples having low, normal and high fibrinogen levels. The present invention thus provides a method and apparatus for accurately determining the fibrinogen level of a patient test sample with greater accuracy than prior art methods.